Methyltransferases (MTs) are a diverse class of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a substrate molecule. MTs catalyze reversible covalent modifications of proteins as well as a variety of biological molecules, including proteins, such as histones, as well as DNA, RNA, hormones, lipids and metabolites. Over 200 distinct MTs are predicted from the human genome and over 60 MTs have been linked to disease states including cancer, neurodegenerative disorders and many others. Because of the important role MTs play in disease, there is a large demand for simple MT assays for use in high-throughput screening (HTS) of small molecule libraries to identify compounds that can modulate MT activity. Unfortunately, current methods for assaying MT activity are not optimal for HTS because they are not homogeneous and often not generalizable to screening all of the different MT classes. A more straightforward MT assay would be to directly measure the reactants and/or products of MT enzymatic activity in a homogeneous format. All MTs utilize SAM as the methyl-donating compound and the invariable byproduct of this reaction is S-adenosylhomocysteine (SAH). Therefore, assays that directly detect the disappearance of SAM or the appearance of SAH would be useful for measuring the activity of any MT, regardless of the substrate. Using a patented sensor technology developed by Lucerna, we will develop a universal MT assay based on a simple, homogeneous fluorescence sensor that is highly specific for either SAM or SAH at physiological concentrations of these cofactors. This Phase I SBIR application describes proof-of-concept experiments for developing, characterizing, and optimizing our SAM and SAH sensors in an assay for histone MT (HMT) activity. For Phase II, our assay will be optimized to comply with standards required for HTS and systematically compared to several competing assays to demonstrate the advantages of our technology. PUBLIC HEALTH RELEVANCE: Over 60 methyltransferases (MTs) have been linked to disease states including cancer, neurodegenerative disorders and many others. The extensive role in MTs in human diseases makes them one of the largest and most promising enzymes classes for therapeutic intervention, but the lack of a robust and simple assay currently precludes high throughput screening (HTS) drug discovery. This project will develop a simple, homogenous fluorescence assay that can be applied to any MT, enabling large-scale HTS for MT inhibitors.